The invention relates to recombinant vectors of viral origin which contain a nucleotide sequence coding for a specific polypeptide, and their use for the expression of this polypeptide in muscle cells. The invention also relates to a procedure for producing these vectors, as well as to their uses, in particular as medicines in the field of muscle diseases.
The hitherto unresolved problem of the direct diffusion of a gene towards a specific tissue is an obstacle to the development of gene therapy in the field of muscle diseases.
The various attempts to modify muscle tissue performed hitherto consist mainly of that involving fusion of muscle cells with a host cell (Salminen, A. et al., Hum. Gene Ther. 2, 15-26 (1991); Partridge, T. A. et al., Nature 337, 176-179 (1989), and that involving direct injection of DNA into the muscles (Wolff, J. A. et al., Science 247, 1465-1468 (1991); Acsadi, G., New Biol. 3, 71-81 (1991)).
The method proceeding by the fusion in mice of precursors of muscle cells derived from a normal donor with muscle fibers of a host (Partridge, T. A. et al., mentioned above) has been carried out with success and this cellular therapy has been the subject of preliminary trials in children. However, this approach seems to present too many disadvantages to be applicable to the treatment of muscle diseases. In fact, since the migratory capacities of the precursor cells are reduced to a few millimeters, the cellular implantation of these latter would necessitate millions of injections requiring hours of anaesthesia. Inevitably, there would be the risk of immunological problems leading to rejection phenomena occurring, as in the case of very many grafts. In addition, the treatment of Duchenne""s muscular dystrophy (DMD) not only requires making contact with the skeletal muscles but also with the myocardial cells; the difficulties likely to be encountered in implanting precursors of muscle cells in the myocardium can easily be imagined. Consequently, cellular therapy hardly seems to be appropriate for the treatment of diseased cells showing such dissemination in the organism.
Gene therapy by direct in vivo introduction of nucleic acids into the interior of organs is an attractive method on account of its simplicity, but its development is confronted with a number of obstacles. In particular, the expression of genes in the muscles remains localised at the site of injection (Wolff, J. A. et al., mentioned above) and seems to be of quite limited duration, particularly in cardiac muscle (Acsadi, G. et al., mentioned above).
The aim of the present invention is precisely to make possible the introduction of a very large number of nucleic acids into a considerable number of muscle cells (up to 50% or more) of a human or animal organism, whether these muscle cells be those of skeletal muscle or those of the myocardium.
The present invention relates more particularly to the transport of nucleic acids to target muscle cells by the blood, while protecting these nucleic acids against attack by various blood constituents.
Another aim of the present invention is to make available to the public pharmaceutical compositions which make possible the treatment of muscle diseases, and more particularly genetic diseases of the muscle system, or also diseases, the localization of which in the organism makes them accessible to the expression products of the above-mentioned nucleic acids, these products being secreted by the said muscle cells.
The present invention follows from the discovery made by the inventors of the fact that beta-galactosidase activity is found in many tissues after injection into mice of recombinant vectors of viral origin, more particularly of adenoviral origin, into the genome of which the gene coding for beta-galactosidase has been inserted. Such tissues include the lungs, liver, intestine, heart and the skeletal muscles. The expression of the gene for beta-galactosidase is constant with time, since the proportion of blue-coloured cells (colour obtained subsequent to gene expression) in the muscle tissue is more or less the same from one month to the next.